Enzymes, which are a kind of catalyst produced by organisms, are mainly composed of proteins. Reaction materials of reactions catalyzed by the enzymes are known as substrates. For substantially all the reactions within living organisms, there exist enzymes corresponding thereto; and their enzyme reactions are performed under moderate conditions within the living organisms, thereby contributing to the maintenance of life. Accordingly, since a reduction in the action of an enzyme (enzyme activity) may cause a disease, it is prevalent in clinical tests to diagnose diseases by measuring activities of some kinds of enzymes in blood and urine.
Also, enzyme reactions are highly substrate-specific and are widely used as an energy-saving industrial process. For example, in fat and oil chemical industry, lipase is used for decomposing fats and oils. Lipase is an enzyme which hydrolyzes glycerol esters (triglycerides) of long-chain fatty acids. Lipase within alimentary canals mainly derives from the pancreas, though part thereof is secreted by the stomach and intestines. Lipase activity in blood has been known to rise in pancreatic diseases. Accordingly, the lipase activity in blood is measured to evaluate pancreatic diseases.
Measuring an enzyme reaction includes measuring quantitative and temporal changes in the substrate, which is a reaction material of the reaction catalyzed by the enzyme, and in a product generated by the reaction. Namely, the greater the ratio by which the substrate changes into the product, the higher the enzyme activity becomes. On the other hand, the smaller the ratio is, the lower the enzyme activity becomes. Also, when the change from the substrate to the product is measured over time, the reaction speed of the enzyme reaction can be analyzed.
Conventionally, in methods of measuring quantitative changes in the substrate and product, differences in physical or chemical properties of the substrate and product are utilized, so as to measure their respective quantitative changes. Examples thereof include: (1) a method in which the substrate or product is specifically caused to develop a color after the termination of the enzyme reaction, and is subjected to colorimetric determination, whereby the amount of increase or decrease in the substrate or product is determined (colorimetric determination method); (2) a method in which a compound labeled with a radioisotope (radioactive compound) is used as a substrate, the fact that the product generated by the enzyme reaction becomes a different radioactive compound is used to separate the radioactive compounds from each other, and the quantity of radioactivity is measured, whereby the amount of increase or decrease in the substrate or product is determined; (3) a fatty acid generated by the enzyme reaction is neutralization-titrated after the termination of the enzyme reaction (titration method); (4) a method in which the substrate is suspended in water, and the change in turbidity of the substrate solution upon decomposition of the substrate by the enzyme reaction is measured, whereby the amount of increase or decrease in the substrate or product is determined (turbidity measurement method); (5) a method in which, by utilizing spectroscopic differences between the substrate and product, wavelengths of light specifically absorbed by their respective materials (absorption wavelengths) are used, whereby the amount of increase or decrease in the substrate or product is determined from change in absorbance (spectroscopic measurement method); and the like. Among them, in particular, the spectroscopic measurement method and turbidity measurement method can continuously measure the enzyme reaction.
The above-mentioned measuring methods respectively have the following problems, however. First, in the colorimetric determination method, a color-producing reagent must react with the substrate or product to specifically color the same after the termination of the enzyme reaction. This complicates the operation and taking time for measurement, and the enzyme reaction cannot be measured continuously. Further, when the substrate is an aqueous suspension, it is difficult to transmit light therethrough, thus being problematic in that measurement cannot be fully performed.
In the method using the radioisotope, since the substrate or the product is separated after the termination of the enzyme reaction so as to measure the quantity of radioactivity, the operation is complicated, the measurement takes time, and the enzyme reaction cannot be measured continuously. Also, since the radioisotope is used, security and restriction are problems on the site of use.
Though the substrate and the product can be measured without separation after the termination of the enzyme reaction in the titration method, it is problematic in that the enzyme reaction cannot be measured continuously.
Though the turbidity measurement method is easy to operate and can continuously measure the enzyme reaction, the light scattered from the suspended substrate is measured and not the substrate itself. Since the enzyme reaction itself is not observed, this method is problematic in terms of reliability.
By contrast, the spectroscopic measurement method is a useful means in that the enzyme reaction can be measured easily and continuously without separating the substrate or product. Nevertheless, in the case where the solution to be measured has a turbidity, when the substrate is not water-soluble in particular, it is problematic in that quantitative measurement is difficult. It has not conventionally been applied to a turbid sample in such an aqueous solution. The influence of the above-mentioned turbidity is strong in particular when the measurement wavelength is within the range from ultraviolet to visible. Though the turbidity becomes less influential when the measurement wavelength is that of infrared light, it has been known that, since the absorption of water as a solvent is so much in the infrared range, it is necessary to use a cell having a thickness on the order of several hundred microns or use total reflection absorption method (Jiro Hiraishi ed., "Furie Henkan Sekigaibunkoho (Fourier Transform Infrared Spectroscopy)," Gakkai Shuppan Center, (1985), pp. 163-171).
When the spectroscopic measurement method is applied to an enzyme reaction, strict stirring is necessary due to the dependence of the enzyme reaction on the stirring speed and the like. In the above-mentioned cell having a thickness on the order of several hundred microns, however, stirring has been impossible. Further, typical measuring apparatus for the total reflection absorption method have failed to correctly measure enzyme activity since stirring cannot be effected therein.
In order to eliminate the above-mentioned essential problems resulting from spectroscopic measurement method, it is an object of the present invention to provide an enzyme reaction measuring method and its measuring apparatus in which it is unnecessary for the substrate and the product to be separated from each other, operation is easy, enzyme reactions can be measured continuously, and enzyme reactions can be measured with a high reliability.